High-affinity monoclonal antibodies for botulinum toxin type b

ABSTRACT

High affinity antibodies for binding epitopes of BoNT/B and hybridomas that produce such antibodies are described. The antibodies may be used in a kit for detecting BoNT/B in a sample.

RELATED APPLICATIONS

This application is claims priority to U.S. Provisional PatentApplication Ser. No. 61/388,477, filed Sep. 30, 2010 the contents ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to high affinity monoclonal antibodies(Mab's) that bind to heavy and light chains of Clostridium botulinumneurotoxin (BoNT) and the associated use of these Mab's in detectingClostridium botulinum.

BACKGROUND OF THE INVENTION

Foodborne botulism is a serious condition in which the patientexperiences a gradual flaccid paralysis, 18 to 36 hours followingconsumption of contaminated food. If untreated, botulism can be fatal.Treatment is a lengthy process that may require hospitalization forseveral months with continuous mechanical ventilation (CDC, 1998, Dembeket al, 2007).

Botulinum neurotoxins (BoNTs) are the causative agents of botulism, andare the most potent naturally-occurring toxins known (Lamanna, 1959).There are seven serotypes of BoNTs, designated A through G, withserotypes A, B, E and F most frequently associated with human cases ofbotulism (Hatheway, 1990). BoNT/A is the most widely studied and bestcharacterized of the BoNT serotypes—a cursory survey of the scientificliterature indicates that there are approximately three times as manypublications about BoNT/A than the next most frequent serotype, BoNT/B.

In the United States from 2001 to 2007, a total of 139 cases offoodborne botulism were reported to the Centers for Disease Control andPrevention (CDC). The majority of these cases were caused byintoxication by BoNT/A (76 cases) or BoNT/E (46 cases), with only 10cases directly linked to consumption of food contaminated with BoNT/B.However, in the same seven years, BoNT/B was the causative agent of 387of the 663 cases of infant botulism (58.4%) recorded by the CDC(National Botulism Surveillance, 2001-2007).

Although BoNT/B is a less frequently observed cause of foodbornebotulism, it is nonetheless a significant threat to food safety. Thelargest recorded outbreaks of foodborne botulism to occur in both theUnited States and United Kingdom (UK) were attributed to the consumptionof food contaminated with BoNT/B. In April 1977 in Michigan, a total of59 patients were diagnosed with type B botulism, caused by eating asauce made from improperly home-canned jalapenos. Eleven of the patientsrequired hospitalization, although there were no reported deaths(Terranova et al., 1978). In June 1989 in the UK, 27 patients wereintoxicated (one of whom died) by BoNT/B-contaminated hazelnut yoghurt(O'Mahony et al., 1990).

At the molecular level, BoNT/A and BoNT/B function in a similar manner.Both toxins are comprised of a 100 kDa heavy chain (Hc) and a 50 kDalight chain (Lc), linked by a single disulphide bond. The Hc functionsby binding nerve cells and facilitates the internalization of the Lc, azinc metalloprotease, into the pre-synaptic neuron at the neuromuscularjunction (Montecucco & Schiavo, 1994; Simpson 2004). The Lc of BoNT/Acleaves synaptosomal-associated protein 25 (SNAP-25) whereas the Lc ofBoNT/B cleaves synaptobrevin-2 (Schiavo et al., 1992; Blasi et al.,1993). Either cleavage event prevents the docking ofacetylcholine-carrying vesicles with the presynaptic membrane, thusblocking the release of the neurotransmitter into the neuromuscularjunction and ultimately prohibiting the contraction of the muscle(Simpson, 2004).

The development of a sensitive sandwich ELISA for the detection ofBoNT/A, with a detection limit of 2 pg/mL was recently reported (Stankeret al., 2008). The mAbs (F1-2, F1-5 and F1-40) that form the foundationof this sandwich ELISA have been extensively characterized. Binding ofthese antibodies to the other serotypes of BoNT was undetectable(Stanker et al., 2008; Scotcher et al., 2009a & b). Whilst these studieshave allowed the development of a test specific for BoNT/A, it is nownecessary to develop a novel collection of mAbs to facilitate thedevelopment of a sandwich ELISA-based test specific to BoNT/B.

SUMMARY OF THE INVENTION

Herein is described the production and characterization of a collectionof monoclonal antibodies (Mab's) specific to BoNT/B.

An embodiment of the invention is the development of a new sandwichELISA, capable of detecting BoNT/B in buffer at concentrationsundetectable by the mouse bioassay.

Another embodiment is the use of the sandwich ELISA to recover BoNT/Bfrom spiked milk samples with minimal sample preparation ormodification.

A further embodiment of the invention is the use of the monoclonalantibodies for in vivo treatment of exposure or infection to Clostridiumbotulinum neurotoxin (BoNT/B) or to serve as a vaccine or therapeuticagent wherein protection may be afforded via administration of theantibodies to those at risk of exposure or wherein infection or presenceof the toxin within the organism has been detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the transmembrane domain and receptorbinding domain of BONT/B.

FIGS. 2A & 2B show the Western Blot of Mab's for the light chain—F24-1,F27-33; and heavy chain receptor domain mABs F26-16, F29-40, MCS6-27 MCS90-1-5-1, MCS 90-21-9-2, MCS 92-23-23-7, and MCS 92-32-10-1.

FIG. 3 is a graph of a luminescent assay of the binding of the Mab's tothe seven serotypes of Clostridium Botulinum.

FIG. 4 is a graph of the effects of SDS concentration and pH on BoNT/Bcapture.

FIGS. 5A & 5B are graphs of Sandwich ELISA for detection of BoNT/B.Panel A, MAb MCS6-27 was used as capture antibody, Biotin labeled mABMCS 92-32, 1-10 was used in conjunction with an HRP-conjugatedstreptavidin as detector. Panel B. mAb MCS6-27 was used as captureantibody and a polyclonal rabbit anti-toxin antibody used as thedetector antibody.

FIG. 6 is a graph of chemiluminescent Sandwich ELSA using MCS6-27 ascapture antibody and biotin labeled mAb MCS 92-32-10-1 as detectorantibody in conjunction with HdRP-conjugated Ruthinium as detector.

STATEMENT OF DEPOSIT

Monoclonal antibodies (Mab) to Clostridium botulinum neurotoxin weredeposited May 5, 2011 under terms of the Budapest Treaty with theAmerican Tissue Culture Collection (ATCC) P.O. Box 1549, Manassas, Va.,20108, USA. The Mab MCS 6-27-1-7 is produced by the hybridoma depositedunder American Type Culture Collection (ATCC) Accession No. PTA-11871and recognizes BoNT/B and BoNT/B Hc. Mab MCS 92-32-1-10 is produced bythe hybridoma deposited under American Type Culture Collection (ATCC)Accession No. PTA-11872 and recognizes BoNT/B and BoNT/B Hc Themicroorganism deposit was made under the provisions of the “BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure”. All restrictions on theavailability to the public of these deposited microorganisms will beirrevocably removed upon issuance of a United States patent based onthis application. For the purposes of this invention, any Mab having theidentifying characteristics of PTA-11871 and PTA-11872 includingsubcultures and variants thereof which have the identifyingcharacteristics and activity as described herein are included.

DESCRIPTION OF THE INVENTION

The terminology used in the description of the invention herein is fordescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description of the inventionand the appended claims, the singular forms “a,” “an,” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurement.

Clostridium botulinum, an anaerobic spore-forming bacterium, produces afamily of botulinum neurotoxins (BoNT, EC 3.4.24.69) [Gill, M.Microbiol. Rev. 46:86-94 (1982)] consisting of seven serotypes, A-G(BoNT/A-BoNT/G). These are considered the most toxic proteins known.Serotype A is synthesized as a single 1,296 amino acid polypeptide,150,000˜Daltons (Da) that is then cleaved endogenously or exogenouslyforming a dichain molecule comprised of an ˜100 kDa heavy chain (Hc) andan ˜50 kDa light chain (Lc) linked by a single disulfide bond[Montecucco, C, and Schiavo, G. Structure and function of tetanus andbotulinum neurotoxins. Quarterly Rev. Biophys. 28:423-472 (1995)]. TheHC mediates toxin entry into neurons, and the Lc functions as azinc-dependent endoprotease cleaving SNAR proteins involved inacetylcholine release resulting in muscular paralysis [Turton, K.,Chaddock, J. A., Acharya, K. R. Trends Biochem. Sci. 27:552-558 (2002)].The crystal structure of BoNT/A was determined at 3.3 Å resolution[Lacyt, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R., and Stevens, R.C. Crystal structure of botulinum neurotoxin type A and implications fortoxicity Nature Structural Biol. 5:898-902 (1998)].

An embodiment of the invention describes high affinity monoclonalantibodies (Mab's) to heavy and light chains of Clostridium botulinumneurotoxin B. The antibodies are IgG₁ subclass Mab's with kappa lightchains that specifically bind BoNT serotype B (BoNT/B). Furthercharacterization of these Mab's and their application to rapidimmunoassay formats is presented.

A further embodiment of the invention describes the use of the Mab's ina test kit for the detection of Clostridium botulinum. Using the abovetest, toxin can be detected in amounts less than that detected by themouse bioassay.

The term “antibody” (Ab) or “monoclonal antibody” (Mab) as used hereinis meant to include intact molecules as well as fragments thereof (suchas, for example, Fab and F(ab′).sub.2 fragments) which are capable ofbinding. The language “monoclonal antibody” is art-recognizedterminology. The monoclonal antibodies of the present invention can beprepared using classical cloning and cell fusion techniques. Theimmunogen (antigen) of interest, e.g. intact 150 kDa toxin, separatedheavy or light chains of BoNT/A, or fragments of the Hc and Lc generatedusing recombinant methods expressed as toxin fragments or toxinfragments fusion proteins, is typically administered (e.g.intraperitoneal injection to wild-type mice or transgenic mice whichproduce desired antibodies, such as human antibodies, rats, rabbits orother animal species which can produce native or human antibodies. Theimmunogen can be administered alone or as a fusion protein to induce animmune response. Fusion proteins comprise the peptide against which animmune response is desired coupled to a carrier protein, such as.beta.-galactosidase, glutathione S-transferase, keyhole limpethemocyanin (KLH), and bovine serum albumin, to name a few. In thesecases, the peptides serve as haptens with the carrier proteins. Afterthe animal is boosted, for example, three or four times, the spleen isremoved and splenocytes are extracted and fused with myeloma cells usingthe well-known processes of Kohler and Milstein (Nature 256: 495-497(1975)) and Harlow and Lane (Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory, New York 1988)). The resulting hybrid cellsare then cloned in the conventional manner, e.g. using limitingdilution, screened and the resulting positive clones, which produce thedesired monoclonal antibodies, cultured.

Antibodies, or fragments thereof, may be labeled using any of a varietyof labels and methods of labeling. Examples of types of labels which canbe used in the present invention include, but are not limited to, enzymelabels, radioisotopic labels, non-radioactive isotopic labels,chromogenic labels, fluorescent labels, and chemiluminescent labels[Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988) 555-612].

The present invention still further pertains to a method for detectingBoNT/B in a sample containing BoNT/B. The method includes contacting thesample with an antibody which binds an epitope of BoNT/B, allowing theantibody to bind to BoNT/B to form an immunological complex, anddetecting the formation of the immunological complex and correlatingpresence or absence of the immunological complex with presence orabsence of BoNT/B in the sample. The sample can be biological,environmental or a food sample.

The language “detecting the formation of the immunological complex” isintended to include discovery of the presence or absence of BoNT/B in asample. The presence or absence of BoNT/B can be detected using animmunoassay. A number of immunoassays used to detect and/or quantitateantigens are well known to those of ordinary skill in the art. SeeHarlow and Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988) 555-612. Such immunoassays include antibodycapture assays, antigen capture assays, two-antibody sandwich assays,and immunoaffinity assays. These assays are commonly used by those ofordinary skill in the art. In an antibody capture assay, the antigen isattached to a solid support, and labeled antibody is allowed to bind.After washing, the assay is quantitated by measuring the amount ofantibody retained on the solid support.

A variation of this assay is a competitive ELISA—as represented by anembodiment of the invention—wherein the antigen is bound to the solidsupport and two solutions containing antibodies which bind the antigen,for example, serum from a BoNT/B vaccine, and a monoclonal antibody ofthe present invention, are allowed to compete for binding of theantigen. The amount of monoclonal bound is then measured, and adetermination is made whether the serum contains anti BoNT/B antibodieswherein detection of large amounts of monoclonal antibody indicates asmall to no antibody against BoNT/A in the serum. This competitive ELISAcan be used to predict immunity in a vaccine following vaccination.

Recombinant GST-fusion peptides of BoNT/A for the successfulidentification of the epitope sites of mAbs F1-2, F1-5 and F1-40, highaffinity mAbs that bind BoNT/A were previously used. These studiesdemonstrated that recombinant peptides of BoNT/A could be successfullyemployed as surrogates for the BoNT/A holotoxin (Scotcher et al., 2009a& b). Based upon this observation, three recombinant GST-BoNT/B fusionpeptides (Lc, H1, H5) were produced for immunizing mice in order toproduce mAbs that bind wild type, intact BoNT/B. It was reasoned thatthe fusion peptides would not be toxic to the mice, and also would allowthe production and identification of mAbs that bind specifically to thelight chain (Lc), to the transmembrane domain (H1) and to thereceptor-binding domain (H5) of BoNT/B (see FIG. 1).

Two mAbs that bound BoNT/B Lc (F24-1 and F27-33) and seven mAbs thatbound the receptor-binding domain of the Hc (F26-16, F29-40, MCS90-1-5-1, MCS 90-21-9-2, MCS 92-93-237, MCS 92-32-10-1 and MCS6-27) aredisclosed. No mAbs that bound the transmembrane domain of Hc wereidentified. The Hc of mAb F26-16 was found to be an IgA isotype, whereasthe Hc of the other eight mAbs were IgG isotypes. All nine mAbsexhibited kappa light chains.

Binding of each mAb to the collection of BoNT/B GST-fusion peptidesshown in FIG. 1 was investigated by direct-binding ELISA. Each purifiedmAb bound the GST-fusion peptide used as an antigen to produce it, butnot to any other GST-fusion peptide of BoNT/B. By using smallerGST-fusion peptides, the epitope location for each mAb could belocalized to several hundred amino acids.

Both mAbs F24-1 and F27-33 bound the Lc of BoNT/B between residues A212and K441. MAbs F26-16 and F1-40 bound the receptor-binding domain of theHc, between E1082 and E1291. The epitope of mAb MCS6-27 could not befurther defined because it failed to bind either smaller sub-peptides(H2, H3) of the receptor-binding domain (H5). Since peptides H2 and H3divide the receptor-binding domain, we speculated that mAb MCS6-27 mightbind a region spanning between these two peptides. To test thishypothesis, we constructed a small synthetic peptide 20-mer(EERYKIQSYSEYLKDFWGNP) (SEQ ID NO: 56) corresponding to the amino acidsequence spanning peptides H2 and H3. However, no binding to thispeptide was observed (data not shown). It is possible that MCS6-27 bindsa discontinous conformational epitope that is not entirely present onpeptides H2 or H3, or the short synthetic peptide tested. It can only beconcluded that the epitope for MCS6-27 lies between amino acids E859 andE1291 of BoNT/B. MAbs MCS 90-1-5-1, MCS 90-21-9-2, MCS 92-93-237, MCS92-32-10-1 bind the HC recombinant peptide HcP5 corresponding to thereceptor binding domain.

Clustal W2 alignment amino acid sequence of the variable regions ofanti-BoNT/B monoclonal antibodies are set forth in Table 3. Joiningsegments indicated with box. Constant region of kappa light chain and C1region of heavy chain in bold. The binding of the nine mAbs to theBoNT/B holotoxin was confirmed by Western blot analysis shown in FIG. 2.All nine mAbs bound the intact BoNT/B holotoxin, with F24-1 and F27-33selectively binding the Lc of reduced BoNT/B, and F26-16, F29-40 andMCS6-27, MCS 90-1-5-1, MCS 90-21-9-2, MCS 92-93-237, and MCS 92-32-10-1binding the Hc of reduced BoNT/B. In order to visualize binding ofMCS6-27 to the nitrocellulose-immobilized BoNT/B, the exposure time wasincreased to two hours, six times greater than that required for theother mAbs. This observation suggests that MCS6-27 binds poorly to theBoNT/B under these conditions, possibly becausenitrocellulose-immobilized BoNT/B is in a conformation that is notoptimal for the binding of mAb MCS6-27.

All of the mAbs except F24-1 bound only to BoNT/B. In contrast, mAbF24-1 bound to plate-immobilized BoNT/G at approximately 20% theintensity of binding to BoNT/B (FIG. 3). A comparison between the BoNT/BLc between A212 and K441, and the corresponding region of the Lc ofBoNT/G (Campbell et al., 1993) revealed 64% identity and 92% similarityin the amino acid sequence, suggesting that some identical or similarresidues that comprise the epitope in BoNT/B are also present in BoNT/G.

The mAbs identified via the two different screening strategies displayedmarkedly different properties. None of the mAbs identified via thetraditional direct-binding ELISA were able to capture BoNT/B fromsolution in the absence of SDS. Conversely, MCS 6-27, MCS 90-1-5-1, MCS90-21-9-2, MCS 92-93-237, MCS 92-32-10-1 the only mABs identified viathe capture-capture ELISA, were found to be excellent capture antibodiesin the absence of SDS. Furthermore, a prophylactic, intravenous (iv)injection of 40 μg of mAb MCS6-27, 2.5 μg of mAb MCS 90-1-5-1, 40 μg ofmAb MCS 90-21-9-2, 0.625 μg of MCS 92-23-23-7 and 40 μg of MCS92-32-10-1 per mouse protected 100% of the mice studied from death orany symptoms of intoxication from an iv injection of 460 pg (100 mouseiv LD₅₀) of BoNT/B, consistent with the observation that these couldbind BoNT/B in vitro under physiological conditions. The ratio ofantibody to LD₅₀ units of BoNT/B toxin neutralized ranged from 0.2 to0.003 μg mAb per 1 LD₅₀ unit. A study into the neutralization of BoNT/Ain mice using mAb F1-2 revealed a ratio of 0.14 μg F1-2 per 1 LD₅₀ unitof BoNT/A (Cheng et al., 2009). It is possible that a lower quantity ofmAb MCS6-27 and MCS 92-32-10-1 are sufficient to neutralize 1 LD₅₀ unitof BoNT/B, and is the subject of future studies. We hypothesize thatsince mAbs F24-1, F26-16, F27-33 and F29-40 were unable to bind BoNT/Bin physiological buffer, they did not bind toxin in vivo and thus failedto protect mice from the neurotoxic effects of BoNT/B. The ability of amAb to capture antigen from solution appears to be an indicator fortoxin neutralization potential. Our data suggest that a capture-capturescreen would be more appropriate than a direct binding ELISA if mAbsthat neutralize toxin are to be identified.

The observation that all of the mAbs isolated using the direct bindingELISA screen gave strong ELISA titration curves but failed to bind toxinfrom solution suggested to us that immobilization of toxin on themicrotiter plates results in an alteration of some physicochemicalproperties (e.g., surface charge or tertiary structure) so that acryptic epitope is exposed that is not available when the toxin is insolution under physiological conditions. MAb MCS 6-27, MCS 90-1-5-1, MCS90-21-9-2, MCS 92-23-23-7, MCS 92-32-10-1 all of which captured toxin insolution and protected from toxin exposure in vivo must bind surfaceepitopes not altered by immobilization in the wells of the microtiterplate. In an effort to clarify this possibility, we investigated theeffects of two factors, the pH and SDS concentration of the capturebuffer, on the ability of five of the mAbs to capture BoNT/B fromsolution. The effect of altering the pH was less marked than that ofSDS, although both Lc-binding mAbs (F24-1, F27-33) displayed a greatersensitivity to alkaline pH than the other three Hc-binding mAbs (F26-16,F29-40 and MCS6-27). It is possible that the increasingly basicconditions greater than pH 6.0 affected the mAb or the toxin Lc in amanner that caused decreased binding. The same pH conditions did notaffect the toxin Hc, or Hc-binding mAbs, in a way that decreasedbinding. However, in the absence of experiments that assay the structureof both antibody and toxin at each pH, this eventuality cannot bedetermined.

The concentration of SDS in the capture buffer had a dramatic effect onthe ability of the mAbs to bind toxin in solution. SDS is often used atconcentrations of 0.1% (˜3.5 mM) in acrylamide gels and associatedbuffers to bind proteins and cause major conformational changes,commonly known as denaturation. However, there exists a dynamic range ofSDS concentration at which SDS binds the protein and inducesconformational changes that do not completely denature the protein. Atlower concentrations, SDS monomers bind to certain high energy sites onthe protein. As the concentration of SDS increases, SDS monomers bind ina cooperative manner ultimately resulting in the saturation of binding.(Robinson & Tanford, 1975; Bhuyan, 2009). It has been shown that theminimum concentration at which SDS can affect protein conformation is0.1 mM, whereas some proteins can become 100% denatured at SDSconcentrations of 1 mM (Reynolds & Tanford, 1970, Miyazawa et al.,1984). Thus, the effects SDS concentrations up to 1 mM on antibodycapture were investigated.

Over the range of SDS concentrations investigated, the capture mAbsseparated into two distinct groups, again mirroring the screen by whicheach mAb was identified. MAb MCS6-27 captured BoNT/B at SDSconcentrations between 0 and 0.4 mM, whereas the mAbs F24-1, F27-33,F26-11 and F29-40 optimally captured toxin at SDS concentrations between0.5 and 0.9 mM. A boundary between the two mAb populations fell at0.4-0.5 mM SDS. We hypothesize that across the range of SDSconcentrations from 0 to 1.0 mM, the SDS altered the proteinconformation of either BoNT/B or the capture mAb in a manner thatfacilitated or inhibited binding. It is unlikely that either BoNT/B orMCS6-27 were completely denatured at 0.5 mM SDS, as the concentration ofSDS seems too low to have that effect. However it is possible that theconformation of one or both proteins was altered sufficiently, or thatkey high energy sites on one or both proteins were blocked by monomericSDS, resulting in the abolition of binding.

MAbs F24-1, F26-16, F27-33, F29-40, MCS6-27 MCS 92-23-23-7, MCS92-32-10-1, MCS 90-1-5-1 and MCS 90-21-21-2 and the anti-BoNT/B rabbitand/or horse polyclonal were used in all combinations to identifycapture and detector pairs for the development of a sandwich ELISA forBoNT/B detection. Using mAb F24-1 for capture and biotinylated F29-40for detection, an SDS-dependent sandwich ELISA was constructed. However,the L.O.D. for this assay was approximately 90 pg/mL BoNT/B, almost twoorders of magnitude higher than the L.O.D. of 1 pg/mL observed for themost sensitive sandwich ELISA that, under physiological conditions, usedmAb MCS6-27 for capture and the anti-BoNT/B rabbit polyclonal antiserafor detection. The L.O.Q. was determined to be approximately 2 pg/mL.Since 100 uL of sample is evaluated in the sandwich ELISA describedhere, the L.O.D. by mass of BoNT/B was 100 fg. The mouse LD₅₀ of theBoNT/B preparations used in our laboratory, when injectedinterperitoneally was found to be 8-10 pg per mouse (20 pg/mL). Thissandwich ELISA is therefore approximately 50-fold more sensitive thanthe mouse bioassay for the detection of BoNT/B.

Monoclonal antibodies MCS 6-27, MCS 90-1-5-1, MCS 90-21-9-2, MCS92-23-23-7, MCS 92-32-10-1 were evaluated to identifymonoclonal-monoclonal capture detector pairs for development of asandwich ELISA. An advantage of a monoclonal-monoclonal pair versus amonoclonal-polyclonal pair is the unlimited availability of highlyconsistent monoclonal antibodies for an assay ensuring greater assaystability and reproducibility over time. Using mAb MCS 6-27 as captureantibody and mAb biotinylated MCS 92-32-10-1 as detector anSDS-independent sandwich ELISA was constructed with an L.O.D. of 23pg/mL (approximately equivalent to one mouse LD₅₀). The greaterstability and reproducibility of a mAb/mAb sandwich ELISA make this amore desirable assay than the mAb/polyclonal assay described above.

We have previously described the development and application of asandwich ELISA for the detection of BoNT/A in skim, 2% and whole milk.It was shown that good recoveries were observed when samples were spikedwith as little as 312 pg/mL BoNT/A, but that defatting by centrifugation(14,000×g, 15 min, 6° C.) or a 10-fold dilution step was necessary tominimize sample interference (Stanker et al., 2008).

The monoclonal/polyclonal assay sandwich described here for thedetection of BoNT/B in milk (skim, 2% and whole) does not require anydefatting or dilution step. Toxin was readily detectable at aconcentration of 39 pg/mL (3.9 pg by mass) in all three milk types, withrecoveries of greater than 80%. Although the human oral LD₅₀ for BoNT/Bhas not been determined, the estimated human oral lethal dose (LD) forBoNT/A is 1 μg/kg body weight, or 70 μg for the average-sized human of70 kg (Amon et al., 2001). Assuming a similar toxicity for BoNT/B, thesandwich ELISA can detect as little as ˜ 1/1,800,000 of the human LD₅₀.Similarly, using the MCS 6-27/MCS92-32-10-1 sandwich ELISA BoNT/B wasreadily detected in milk without the need for defatting or other samplepreparation. Toxin was readily detected in whole milk at a concentrationof 2.4 pg/mL with recoveries greater than 104%. Thus, the mAb/mAbsandwich ELISA can detect as little as 1/1,000,000 of the human LD.

In an antigen capture assay, the antigen is attached to a solid support,and labeled antibody is allowed to bind. The unbound reactants areremoved by washing, and the assay is quantitated by measuring the amountof antigen that is bound. In a two-antibody sandwich assay, one antibodyis bound to a solid support, and the antigen is allowed to bind to thisfirst antibody. The assay is quantitated by measuring the amount of alabeled second antibody that can bind to the antigen. These immunoassaystypically rely on labeled antigens, antibodies, or secondary reagentsfor detection. These proteins can be labeled with radioactive compounds,enzymes, biotin, or fluorochromes. Of these, radioactive labeling can beused for almost all types of assays and with most variations.Enzyme-conjugated labels are particularly useful when radioactivity mustbe avoided or when quick results are needed. Biotin-coupled reagentsusually are detected with labeled streptavidin. Streptavidin bindstightly and quickly to biotin and can be labeled with radioisotopes,enzymes or other reporter molecules such as microdots, nanoparticles,fluorochromes or electrochemical tags. Fluorochromes, although requiringexpensive equipment for their use, provide a very sensitive method ofdetection. Antibodies useful in these assays include monoclonalantibodies, polyclonal antibodies, and affinity purified polyclonalantibodies. Those of ordinary skill in the art will know of othersuitable labels which may be employed in accordance with the presentinvention. The binding of these labels to antibodies or fragmentsthereof can be accomplished using standard techniques commonly known tothose of ordinary skill in the art. Typical techniques are described byKennedy, J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), Schurs, A. H.W. M., et al. 1977 (Clin. Chim Acta 81:1-40), Bobrovnik, S. A. 2003 (J.Biochem. Biochys. Methods 57:213-236), and Friguet et al 1985 (J.Immunol. Methods 77:305-319).

Examples of suitable enzyme labels include malate hydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, betagalactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels include .sup.3 H, .sup.125 I,.sup.131 I, .sup.32 P, .sup.35 S, .sup.14 C, .sup.51 Cr, .sup.57 To,.sup.58 Co, .sup.59 Fe, .sup.75 Se, .sup.152 Eu, .sup.90 Y, .sup.67 Cu,.sup.217 Ci, .sup.211 At, .sup.212 Pb, .sup.47 Sc, and .sup.109 Pd.

Examples of suitable fluorescent labels include an .sup.152 Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, a fluorescamine label, etc.

Examples of chemiluminescent substrates include a luminal substrate, anisoluminal substrate, an aromatic acridinium ester substrate, animidazole substrate, an acridinium salt substrate, an oxalate esterlabel, a luciferin substrate, a luciferase label, an aequorin label,etc.

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythese materials, or their functional derivatives, are combined inadmixture with a pharmaceutically acceptable carrier vehicle. Suitablevehicles and their formulation, inclusive of other human proteins, e.g.,human serum albumin, are described, for example, in Remington'sPharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa.(1980)). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of the above-described compounds together with asuitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid), agars, agarose, or ethylenevinylacetate copolymers. Alternatively, instead of incorporating theseagents into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate)-microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1980).

Administration of the antibodies disclosed herein may be carried out byany suitable means known to one of skill in the art, includingparenteral injection (such as intraperitoneal, subcutaneous, orintramuscular injection), orally, or by topical application of theantibodies (typically carried in a pharmaceutical formulation) to anairway surface. Topical application of the antibodies to an airwaysurface can be carried out by intranasal administration (e.g., by use ofdropper, swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatus for administering respirableparticles of pharmaceutical formulations are well known, and anyconventional technique can be employed. Oral administration may be inthe form of an ingestible liquid or solid formulation.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1 or more separate doses, followed by other doses given atsubsequent time intervals required to maintain and or reinforce theresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitable treatmentschedules include: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedulessufficient to elicit the desired responses expected to reduce diseasesymptoms, or reduce severity of disease.

An embodiment of the invention also provides kits which are useful forcarrying out the present invention. The present kits comprise a firstcontainer means containing the above-described antibodies. The kit alsocomprises other container means containing solutions necessary orconvenient for carrying out the invention. The container means can bemade of glass, plastic or foil and can be a vial, bottle, pouch, tube,bag, etc. The kit may also contain written information, such asprocedures for carrying out the present invention or analyticalinformation, such as the amount of reagent contained in the firstcontainer means. The container means may be in another container means,e.g. a box or a bag, along with the written information.

Materials and Methods

Construction, expression and purification of recombinant BoNT/B-GSTfusion proteins

Commercial enzymes (Phusion High-Fidelity DNA Polymerase, BamHI, XhoI,T4 polynucleotide kinase [3′ phosphatase minus], T4 DNA ligase [NewEngland BioLabs, Inc., Bethesda, Md.]) were used according to themanufacturer's recommendation. Primers used were purchased fromIntegrated DNA Technologies (Coralville, Iowa) and are shown in Table 1.Plasmid construction and manipulation was performed in Escherichia coliTOP10 cells (Invitrogen, Carlsbad, Calif.) grown aerobically inLuria-Bertani (LB) medium at 37° C. supplemented with 100 μg/mLampicillin (Miller, 1972). Plasmids or DNA were purified using theQuickClean 5M range of kits (GenScript Corp., Piscataway, N.J.). Allautomated DNA sequencing was performed using the Big Dye TerminatorVersion 3.1 and XTerminator reagents, and a 3730 DNA Analyzer (AppliedBiosystems, Foster City, Calif.).

Total genomic DNA from Clostridium botulinum (Strain Okra/Type B1),generously provided by Eric Johnson (University of Wisconsin, MadisonWis.), was used as a template to amplify the fragments of the light andheavy chains (Lc, L1, L2, Hc, H1, H2, H3, H4, H5,) using the primersindicated (see FIG. 1 and Table 1). Stop codons (TAA) were introducedwhen not present within the genomic DNA of the cloned region. Allsubsequent BoNT/B DNA fragments were cloned into plasmid pCR4-TOPO(Invitrogen) to allow sequencing using primers M13F and M13R. Additionalprimers (B-intseqF, B-intseqR) were required for the longest fragments,Hc and H1. The pCR4-derived plasmids were then digested using BamHI andXhoI, the BoNT/B fragment was purified and ligated into BamHI- andXhoI-digested pGS-21a (Genscript) to yield the correspondently named pGSplasmid (e.g. pGS-H1 for fragment H1). All pGS-21a-derived plasmids weresequenced using primer pGS-F and pGS-R, to confirm the correctintegration of the BoNT/A fragment into the vector. The expression andpurification of all GST-fusion proteins was performed as previouslydescribed (Scotcher et al., 2009a).

The recombinant DNA methods used in this study were approved by theInstitutional Biosafety Committee. DNA sequences determined in thisstudy have been deposited in GenBank and accession numbers are listed inTable 2.

TABLE 1 Primers Primer Sequence Constructs SEQ ID NO: B-LcFGGATCCATGCCAGTTACAATAAATAATTTTAATTATAATG Lc, L1 SEQ ID NO: 40 B-LcRCTCGAG TTATTTAACACTTTTACACATTTGTATCTTATATAC Lc, L2 SEQ ID NO: 41B-LcintF GGATCCGCAAGTATATTTAATAGACG L2 SEQ ID NO: 42 B-LcintR CTCGAGTTAGCCTTTGTTTTCTTGAAC L1 SEQ ID NO: 43 B-HcFGGATCCGCTCCAGGAATATGTATTGATGTTG Hc, H1, H4 SEQ ID NO: 44 B-HcR CTCGAGTTATTCAGTCCACCCTTCATCTTTAG Hc, H3, H5 SEQ ID NO: 45 B-HcintR1 CTCGAGTTAGCTATTATATTTATTAAACATTTC H1 SEQ ID NO: 46 B-HcintF2GGATCCGAAATTTTAAATAATATTATCTTAAATTTAAG H2, H5 SEQ ID NO: 47 B-HcintR2CTCGAG TTAGCTATATGATTGAATTTTATATC H2, H4 SEQ ID NO: 48 B-HcintF3GGATCCGAATATTTAAAAGATTTTTGGGG H3 SEQ ID NO: 49 M13F GTAAAACGACGGCCAGseq. pCR4 SEQ ID NO: 50 plsamids M13R CAGGAAACAGCTATGAC seq. pCR4SEQ ID NO: 51 plasmids B-intseqF CAATAGATAATGCTTTAACTAAAAGAAATGseq. Hc,H1 SEQ ID NO: 52 B-intseqR GTGTTCTATCTATATCACCATC seq. HcSEQ ID NO: 53 pGS-F CAAATTGATAAGTACTTGAAATCC seq. pGS-21a SEQ ID NO: 54plasmids pGS-R GCTAGTTATTGCTCAGAGG seq. pGS-21a SEQ ID NO: 55 plasmids

TABLE 2 Characterization of monoclonal antibodies to BoNT/B Screening HcPeptides Accession # of Lc and Antibody method isotype bound* Epitopelocation Hc sequence F24-1 Direct binding IgG1 Lc, L2 A212-K441Lc-GU799549 Hc-GU799550 F24-4 Direct binding IgG1 n/d n/d n/d F26-16Direct binding IgA Hc, H3, H5 E1082-E1291 Lc-GU799551 Hc-GU799552 F27-33Direct binding IgG1 Lc, L2 A212-K441 Lc-GU799553 Hc-GU799554 F29-38Direct binding IgM n/d n/d n/d F29-40 Direct binding IgG1 Hc, H3, H5E1082-E1291 Lc-GU799555 Hc-GU79556 MCS 6-27 Capture-capture IgG1 Hc, H5E859-E1291 Lc-GU79557 Hc-GU79558 SS 90-21-9-2 Cc IgG1 Hc, H5 E859-E1291SS 92-23-23-7 Cc IgG2b Hc, H5 E859-E1291 SS 92-32-10-1 Cc IgG1 Hc, H5E859-E1291 SS 90-1-5-1 Cc IgG1 Hc, H5 E859-E1291 Cc—Capture-capture

TABLE 3 Clustal W2 alignment amino acid sequence of the variable regionsof anti- BoNT/B monoclonal antibodies. Light Chains (Lc)

Heavy Chains (HC)

Monoclonal Antibody Procedure

The method used for monoclonal antibody production has been describedpreviously (Stanker et al., 2008). Significant differences from thismethod are described below.

Solutions of three peptide fragments at the concentrations indicated(Lc, 90 μg/mL; H1, 186 μg/mL; H5, 265 μg/mL) were mixed with SigmaAdjuvant System #S6322 according to manufacturer's instructions(Sigma-Aldrich, St. Louis, Mo.). Five female BALB/cJ mice (SimonsenLaboratories, Gilroy, Calif.) were immunized three times at 2-weekintervals by intraperitoneal injection (i.p.) of 100 μL of eachantigen-adjuvant solution. Two weeks after the third injection, serumwas obtained from each mouse and evaluated for anti-BoNT/B antibodiesvia direct binding ELISA screens. Mice were injected i.p. with 2 μg ofthe appropriate peptide fragment in 0.01 M phosphate buffered saline(PBS; #P-3813, Sigma-Aldrich) three days prior to being euthanized andsubjected to the fusion procedure.

Supernatants from cell fusion plates were subjected to screening eitherby direct binding ELISA or by capture-capture ELISA screens.

Screening Methods

Direct binding ELISA screens were performed as previously described(Stanker et al., 2008), using microtiter plates coated with 50 μL perwell of a 0.1 μg/mL solution of BoNT/B (Strain Okra/Type B1,Metabiologics Incorporated, Madison Wis.) in 0.05M sodium carbonatebuffer, pH 9.6. Binding was visualized using SuperSignal West DuraExtended Duration Substrate (Pierce, Rockford, Ill.) according tomanufacturer's instructions. The plates were incubated for 3 min at roomtemperature and luminescent counts recorded using a Wallac Victor 2Multilabel Counter (PerkinElmer Inc., Waltham, Mass.).

Capture-capture ELISA screens were performed as follows. Unless statedotherwise, 50 μL per well of all solutions were used. Microtiter plateswere coated with a 1 μg/mL solution of goat anti-mouse IgG Fc gamma#AP127 (Millipore, Billerica Mass.) in 0.05 M sodium carbonate buffer,pH 9.6 overnight at 4° C. The IgG solution was aspirated and non-coatedsites blocked by adding 300 μL per well of 3% non-fat dry milk inTris-buffered saline containing 0.05% Tween-20 (NFDM-TBST) and theplates were incubated for 1 h at 37° C. The plates were washed once with0.05% Tween-20, then cell culture supernatants were added and the plateswere incubated at 37° C. for 1 h. The plates were washed three timeswith 0.05% Tween-20, then a solution of BoNT/B in NFDM-TBST (50 ng/mL)was added and the plates were incubated at 37° C. for 1 h. Plates werewashed three times as before, then a 1 μg/mL solution of anti-BoNT/Brabbit polyclonal antibodies (Metabiologics) in NFDM-TBST was added andthe plates were incubated at 37° C. for 1 h. Plates were washed threetimes as before, then a 1 μg/mL solution of goat anti-rabbitHRP-conjugated polyclonal antibodies #A6154 (Sigma-Aldrich) was addedand the plates were incubated at 37° C. for 1 h. Plates were againwashed three times, and binding was visualized as described above.

Cells from the wells giving positive signals for antibody productionwere cloned by limiting dilution. Hybridomas were then expanded andsmall amounts (usually less than 10 mL) of ascites fluids obtained(Covance Research Products, Inc., Denver, Pa.). Antibodies were purifiedby affinity chromatography on Protein-G (for IgG) or Protein L (for IgA)Sepharose. Bound antibody was eluted with 0.1 M glycine-HCl, pH 2.7.Protein concentrations were determined with a BCA-kit (Pierce) using themicroplate method suggested by the manufacturer.

Antibody Isotyping, Western Blotting, Peptide Binding

All coating, blocking and washing steps of subsequent ELISAs wereperformed as described for the capture-capture ELISA screen, unlessstated otherwise.

The isotype of each antibody was determined using the SBA ClonotypingSystem/HRP in ELISA format, according to manufacturer's instructions(Southern Biotech, Birmingham Ala.).

Western Blotting was performed as previously described (Stanker et al.,2008), except that BoNT/B was used instead of BoNT/A. BoNT/B was reducedby the addition of dithiothreitol (DTT) at a final concentration of 10mM.

Basic characterization of the epitopes of each antibody was performed bydirect binding ELISA. The wells of clear microtiter plates were coatedwith each of the BoNT/B GST-fusion proteins (see FIG. 1), incubatedovernight at 4° C., blocked with NFDM-TBST and washed. Hybridomasupernatant, diluted 1:10 in NFDM-TBST, was added to each well andincubated for 1 h at 37° C. The plate was washed, then a 1 μg/mLsolution of goat anti-mouse HRP-conjugated polyclonal antibodies #A4416(Sigma-Aldrich) was added and the plates were incubated at 37° C. for 1h. Plates were again washed three times, then K-Blue substrate (NeogenCorporation, Lexington, Ky.) was added (100 μL per well) and incubatedwith agitation for 5 min at room temperature. Stop solution (Neogen) wasadded (100 μL per well), and absorbance at 650 nm was measured using aVersaMax microplate reader (Molecular Devices, Sunnyvale Calif.). Eachantibody was tested in triplicate.

Binding of Antibodies to BoNT Serotypes A Through G

Black microtiter plates were coated with the different serotypes ofBoNT, A through G (Metabiologics) for direct binding ELISA analysis asdescribed above. Purified anti-BoNT/B monoclonal antibody at aconcentration of 10 μg/mL in NFDM-TBST was added to each BoNT serotype,and incubated for 1 h at 37° C. The plates were washed, then a 1 μg/mLsolution of goat anti-mouse HRP-conjugated polyclonal antibodies #A4416(Sigma-Aldrich) was added and the plates were incubated at 37° C. for 1h. Plates were again washed three times, and binding was visualizedusing SuperSignal West Dura Extended Duration Substrate as describedabove. Each antibody was assayed against each serotype in triplicate.

Effects of pH and SDS on Capture of BoNT/B from Solution by mAbs

Black microtiter plates were coated with the anti-BoNT/B monoclonalantibodies at 10 μg/mL in 0.05 M sodium carbonate buffer, pH 9.6overnight at 4° C. Non-coated sites were blocked by adding 300 μL perwell of 3% non-fat dry milk in Tris buffered saline containing 0.05%Tween-20. Following incubation for 1 h at 37° C., plates were washedthree times to remove any residual blocking agent. Solutions of BoNT/Bin buffers of various pH and sodium dodecyl sulphate (SDS)concentrations were prepared on a deep-well (2 ml) 96-well plate.Phosphate-buffered saline was added to rows A through G to give a finalpH (±0.1) of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, respectively.SDS was added to columns 1 through 12 to give final concentrations of 0,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0 mM, respectively.The final concentration of BoNT/B was 100 ng/mL in all wells. Thedeep-well plate was gently agitated to mix the solutions, then 100 μL ofeach BoNT/B solution was pipetted into the corresponding well on theblack microtiter plates. Following incubation for 1 h at 37° C., plateswere subsequently treated in an identical manner to the capture-captureELISA described earlier, using the anti-BoNT/B rabbit polyclonalantibodies, goat anti-rabbit HRP-conjugated polyclonal antibodies andSuperSignal West Dura Extended Duration Substrate to allow detection ofBoNT/B capture. Each antibody was assayed in triplicate.

In Vivo Neutralization of BoNT/B

Random groups of 10 mice (4-5 week old female Swiss Webster mice, 19-21g; Charles River Laboratories, Portland Mich.) were injectedintravenously (i.v.) into the lateral tail vein with 100 μL of mAbsdiluted in PBS to a final dosage between 0.156 μg to 40 μg mAbs/mouseone hour prior to administration with 100 μL of BoNT/B holotoxincontaining 1000 pg (200 mouse iv LD₅₀ units) diluted in phosphategelatin buffer. Control mice were treated with 100 μL of PBS instead ofmAbs. Mice were monitored closely over a 14 day period for any symptomsof intoxication, or death. Animal-use protocols were approved by theAnimal Care and Use Committee of the USDA, Western Regional ResearchCenter, Albany, Calif.

Cloning and Sequencing of Monoclonal Antibodies

mRNA coding for the anti-BoNT/B monoclonal antibodies was extracted andpurified from hybridoma cells as previously described (Scotcher et al.,2009a). mRNA was converted to cDNA, and the heavy and light chains ofeach antibody were amplified by PCR as previously described (Wang etal., 2000). The PCR products were gel-purified and treated withpolynucleotide kinase (New England BioLabs). Circularized vector pCR2.1(Invitrogen) was digested with EcoRV and treated with calf intestinalphosphatase (New England BioLabs). The PCR products were ligated intovector pCR2.1, transformed into TOP10 cells, then grown and prepared forDNA sequencing using primers M13F and M13R as described earlier.

Sandwich ELISA

All combinations of mAbs F24-1, F26-16, F27-33, F29-40, MCS6-27, SS90-1-5-1, MCS 90-21-9-2, MCS 92-23-2-7, MCS 92-32-10-1 and theanti-BoNT/B rabbit polyclonal antibody were evaluated as capture anddetector pairs for the development of a sandwich assay for BoNT/Bdetection. Detector mAbs (except the anti-BoNT/B rabbit polyclonal) werebiotinylated using the EZ-Link Micro-Sulfo-NHS-Lc Biotinylation Kit(Thermo Scientific, Rockford Ill.), and then detected using Zymaxstreptavidin-HRP conjugate (Zymed, San Francisco Calif.). Theanti-BoNT/B rabbit polyclonal binding was detected using goatanti-rabbit HRP-conjugated polyclonal described earlier (Sigma).

Three pairs of antibodies were identified: F24-1 (capture) and F29-40(detector); MCS6-27 (capture) and the anti-BoNT/B rabbit polyclonal(detector); MCS6-27 (capture) and MCS 92-32-10-1 (detector). Bindingconditions and solutions were optimized (data not shown) to yield theprotocols described below. Unless stated otherwise, all solution volumeswere 100 μL per well, all incubations were performed for 1 h at 37° C.with gentle agitation, and all washes were performed twelve times inwater plus 0.05% Tween.

White microtiter plates were coated with mAbs F24-1 or MCS6-27 at aconcentration of 5 μg/mL in 0.05 M sodium carbonate buffer, pH 9.6overnight at 4° C. Non-coated sites were blocked by adding 300 μL perwell of 5% non-fat dry milk in Tris buffered saline (pH 8.0) containing0.05% Tween-20 (NFDM-TBST). Plates were incubated and washed asdescribed above. BoNT/B was added to each plate at concentrations from5000 to 0 pg/mL in a two-fold dilution series, in TBS pH 6.0 containing0.6 mM sodium dodecyl sulphate (SDS) for the F24-1-coated plate, or inNFDM-TBST for the MCS6-27-coated plate. Plates were incubated andwashed. Biotinylated mAb F29-40 was added to the F24-1-coated plate at aconcentration of 5 μg/mL in TBS, pH 8.0 containing 0.6 mM SDS. Theanti-BoNT/B rabbit polyclonal detector was diluted 2000-fold inNFDM-TBST and added to the MCS6-27-coated plate. Plates were incubatedand washed. Zymax streptavidin-HRP conjugate was diluted 10,000-fold inTBS, pH 8.0 containing 0.6 mM SDS and added to the F24-1-coated plate.Next, goat anti-rabbit HRP-conjugated polyclonal antibody was diluted2000-fold in NFDM-TBST and added to the MCS6-27-coated plate. Plateswere incubated, washed then binding was visualized as described earlier.

Detection of BoNT/B in Milk Matrices

Both sandwich assays using MCS6-27 (capture) and the anti-BoNT/B rabbitpolyclonal (detector) and MCS6-27 (capture) and MCS 92-32-10-1(detector) were evaluated for their ability to detect BoNT/B in milk.The sandwich ELISA was performed as described above, with the exceptionof the capture stage which was performed as follows.

Three milk types (skim, 2% fat and whole) were spiked with BoNT/B atfinal concentrations of 10000, 5000, 2500, 1250, 625, 312, 156, 78 and39 pg/mL. Several sample treatments were evaluated in order to defat thesamples. Spiked milk samples were centrifuged at 14,000×g for 15 min at6° C. Other samples were simply diluted in two-fold serial dilutions inblocking buffer (Stanker et al., 2008). Following defatting or serialdilution, 100 μL of sample was loaded onto the MCS6-27-coated microtiterplate and incubated for 1 h at 37° C. with gentle agitation. Recovery ofBoNT/B from spiked milk samples is reported as a percentage of therecovery by comparison to a standard curve of BoNT/B spiked intoNFDM-TBST Milk samples were analyzed in triplicate.

Characterization of Anti-BoNT/B Monoclonal Antibodies

A total of nine monoclonal antibodies (mAbs) were identified, cloned andcharacterized (Table 2). Six mAbs (F24-1, F24-4, F26-16, F27-33, F29-38,and F29-40) were identified using a traditional, direct binding ELISAscreening method, and five mAb (MCS 6-27, MCS 90-1-5-1, MCS 90-21-9-2,MCS 92-23-23-7, MCS 92-32-10-1)) was identified using thecapture-capture screen. Isotype analysis revealed that mAb F26-16 was anIgA, F29-38 was an IgM, MCS 92-23 was an IgG2b and the remaining eightmAbs were all IgG1s. All of the mAbs possessed kappa light chains.

Compete sequences of the cloned cDNA coding for the heavy chain variableregion was obtained for six mAbs, a partial sequence was obtained forone mAb, and no Hc sequence we obtained for two antibodies (see FIG. 2).The light chain sequences of of all nine antibodies was obtained.Sequence analysis revealed that mAbs F24-1 and F24-4 have identicalsequences, and most likely represent independent fusions of aclonally-expanded population of lymphocytes. Each mAb possesses uniquevariable region sequences for their heavy and light chains, which can beaccessed online via the Nucleotide Accession Numbers shown in Table 2.The leader sequences, framework regions, complementarity determiningregions (CDRs) and J-regions were identified by inspecting the alignmentof the mAb heavy and light chains to other antibody sequences (Morrison,2002; Wood & Coleclough, 1984; Livesay & Subramaniam, 2004; Scotcher etal., 2009a, b).

Each antibody was studied in Western blot experiments, probing reducedand unreduced 150 kDa BoNT/B holotoxin following separation by SDS-PAGE(FIG. 3). In these experiments, a constant amount (μg) of each mAb wasused to probe the Western blot. Exposure times of Western blots varied(FIG. 3A); mAbs F24-1, F26-16, F27-33 and F29-40 were exposed for 20min, whereas the exposure time for MCS6-27 was 120 min. Constantexposure time of 10 min. was used for MCS 90-1-5-1, MCS 90-21-9-2, MCS92-23-23-7 and MCS 92-32-10-1. All of the mAbs bound the 150 kDa BoNT/Bholotoxin in the unreduced samples. Using reduced samples, mAbs F24-1and F27-33 bound the BoNT/B Lc, and mAbs F26-16, F29-40, MCS6-27, MCS90-1-5-1, MCSS 90-21-9-2, MCS 92-23-23-7 and MCS 92-32-10-1 bound theBoNT/B Hc. Binding to residual 150 kDa BoNT/B holotoxin was alsoobserved in the reduced samples.

In an effort to more precisely define the binding epitopes for thesemAbs, direct binding ELISA experiments were performed to identify whichBoNT/B GST-fusion peptides each antibody bound (See FIG. 1 and Table 2,columns 4 & 5). MAbs F24-1 and F27-33 were both found to bind the Lc andL2 fragments, but not the L1 fragment or any of the other fragmentsderived from the BoNT/B heavy chain. The binding epitope for both mAbsis therefore localized to a 230 amino acid fragment of the BoNT/B lightchain, between amino acids A212 and K441. MAbs F26-16 and F29-40 boundthe Hc, H3 and H5 fragments, but not the Lc, L1, L2, H1, H2 or H4fragments. The binding epitope for both mAbs is therefore localized to a210 amino acid fragment of the BoNT/B heavy chain, between amino acidsE1082 and E1291. In contrast, mAb MCS6-27, MCS 90-1-5-1, MCS 90-21-9-2,MCS 92-23-23-7 and MCS 92-32-10-1 bound fragments Hc and H5, but nobinding to any other fragment was detected, indicating that the bindingepitope is localized to a 433 amino acid fragment of the BoNT/B heavychain, between amino acids E859 and E1291. These observations areconsistent with the binding data obtained from the Western blotexperiments described earlier.

Direct binding ELISAs were performed to test whether each mAb would binduniquely to BoNT/B, or whether binding to other BoNT serotypes could bedetected. These data are summarized in FIG. 4. MAbs F26-16, F27-33,F29-40 and MCS6-27, MCS 90-1-5-1, MCS 90-21-9-2, MCS 92-23-23-7 and MCS92-32-10-1 bound only BoNT serotype B, whereas mAb F24-1 bound BoNTserotype B and serotype G.

Effects of pH and SDS on Capture of BoNT/B in Solution by mAbs

The ability of each mAb to capture BoNT/B from solution was evaluated bysandwich ELISA. Plates were coated with the capture antibody, BoNT/B wasapplied (100 ng/mL in 1×TBS), and capture toxin was then detected usinga rabbit anti-BoNT/B polyclonal antibody followed by an HRP-conjugated,goat anti-rabbit polyclonal antibody (see methods). Initial experimentsindicated that only mAb MCS6-27, MCS 90-21, MCS 92-23, MCS 92-32 wereable to capture BoNT/B from solution. BoNT/B was not detected using mAbsF24-1, F26-16, F27-33 or F29-40 as capture antibodies (data not shown).The effects of the pH and SDS concentration of the capture buffer werethen evaluated for mAbs F24-1, F26-16, F27-33, F29-40 and MCS 6-27.

The effect of pH, ranging from 5.5 to 9.0, on the ability of the mAbs tocapture BoNT/B from solution is summarized in FIG. 5A. For theHc-binding mAbs F26-16, F29-40 and MCS6-27, the pH optimum for BoNT/Bcapture spanned a broad range from pH 6.0 to 8.5. For the Lc-bindingmAbs F24-1 and F27-33, the pH optimum for BoNT/B capture was pH 6.0. Itshould be noted that in the absence of SDS, no BoNT/B captured by mAbsF24-1, F26-16, F27-33 and F29-40 was detected at any pH tested.

FIG. 5B shows the effect of SDS concentration, ranging from 0 to 2.0 mM,on the capture of BoNT/B at the optimal pH for each mAb. MCS6-27captured BoNT/B at SDS concentrations of 0 to 0.4 mM, reaching anoptimal point at 0.4 mM SDS then sharply decreasing such that at SDSconcentrations higher than 0.6 mM, BoNT/B capture was not detected.Conversely, no BoNT/B captured by mAbs F24-1, F26-16, F27-33 and F29-40was detected at 0 mM SDS, but captured BoNT/B was detected withincreasing concentration of SDS up to approximately 0.5 mM. Each ofthese mAbs exhibited the greatest amount of BoNT/B captured at an SDSconcentration between 0.5 and 0.8 mM. At SDS concentrations greater than0.8 mM, the amount of BoNT/B captured declined sharply, with no capturedBoNT/B detectable at SDS concentrations of 2 mM (not shown).

In Vivo Neutralization of BoNT/B

mAbs, F24-1, F26-16, F27-33, F29-40, MCS6-27, MCS 90-1-5-1, MCS90-21-9-2, MCS 92-23-23-7 and MCS 92-32-10-1 were tested individuallyfor their ability to neutralize BoNT/B holotoxin in a systemic mousemodel of intoxication. One hour following intravenous (iv)administration of a mAb against BoNT/B, a lethal dose of BoNT/B (1000pg/mouse or about 100 mouse iv LD₅₀) was delivered iv and the animalsmonitored over time. In the absence of mAbs, intoxicated mice treatedwith PBS alone died within 3.5-5.5 hrs. Mice pre-treated with 80 μg ofF24-1, F26-16, F27-33 or F29-40 were not protected from death and hadsurvival times similar to the PBS treated control mice. In contrast,pre-treatment with 40 μg of MCS6-27 and MCS 92-32-10-1 completelyprotected mice from death as well as any visible symptoms of botulismover the course of 14 days. Pre treatment with 2.5 μg of mAb MCS90-1-5-1 and as little as 0.625 μg of MCS 92-23-23-7 completelyprotected mice.

Sandwich ELISA for BoNT/B Detection

All combinations of mAbs F24-1, F26-16, F27-33, F29-40, MCS6-27, MCS90-21, MCS 92-23, MCS 92-32 and the anti-BoNT/B polyclonal wereevaluated as capture and detector pairs for the development of asandwich assay for BoNT/B detection. The combination of MCS6-27(capture) and rabbit/horse anti-BoNT/B polyclonal (detector) was foundto be most sensitive, as shown in FIG. 6A. This ELISA exhibits a limitof detection (L.O.D., defined as 3 standard deviations above the zero)of approximately 1 pg/mL BoNT/B, and a limit of quantitation (L.O.Q.,defined as 5 standard deviations above the zero) of approximately 2pg/mL BoNT/B. A correlation coefficient (R) value of 0.98 indicates thatthe regression line is an excellent fit. Combination MCS 6-27 (capturemAb) and MCS 92-32 (detector mAb) was the most sensitive mAb pair,L.O.D.˜30 pg/mL, (FIG. 6B). This same antibody pair gave a L.O.D. in aelectrochemiluminescent ELISA or approximately 2 pg/mL (FIG. 6).

Detection of BoNT/B in Milk Matrices

MCS 6-27 and SS 92-32-10-1 as capture/detector were useful as asensitive sandwich ELISA to detect BoNT/B. The L.O.D. and L.O. in asandwich ELISA were 23 pg/mL and 33 pg/mL respectively and the L.O.D ina sandwich ELISA using electrochemiluminescent detection wasapproximately 2 pg/mL. The advantages of a mAb/mAb sandwich ELISA aremany including greater reagent stability and assay reproducibility.Using the same criteria for the MCS 6-27/polyclonal assay, the L.O.D.and L.O.Q. for the mAb/mAb The sandwich ELISA described above was usedto measure BoNT/B toxin recovery from spiked milk samples (skim, 2% andwhole milk). Although several strategies to defat the milk prior to theELISA were evaluated, it was found that no defatting or dilution processwas required for any of the milk types. Recovery of BoNT/B from thespiked milk samples is reported in Table 3. Toxin recovery ranged from80.4% to 106.7% at BoNT/B spike concentrations from 20,000 to 39 pg/mL.An electrochemiluminescent ELISA using MCS 6-27 and MCS 92-32-10-1 ascapture/detector was able to detect BoNT/B equally well in whole milk,and buffer at all concentrations greater than 2.4 pg/mL (the lowestlevel tested).

TABLE 4 Percentage recovery of BoNT/B from milk as determined bymonoclonal/polyclonal sandwich ELISA. Spike level (pg/ml) 20000 50001250 313 156 78 39 Skim milk  98.5 ± 5.7 108.4 ± 4.7 92.3 ± 2.6 100.5 ±6.9  103.2 ± 3.6 94.1 ± 8.5 101.8 ± 13.7  2% milk 106.2 ± 3.5 102.4 ±3.7 101.7 ± 2.0  85.7 ± 2.1 106.7 ± 0.3 86.5 ± 1.6 80.4 ± 12.1 Wholemilk 100.9 ± 3.7  94.0 ± 2.2 99.2 ± 4.5 97.0 ± 3.6 100.6 ± 5.3 84.9 ±4.6 82.5 ± 10.3

-   1. A method for detecting BoNT/A according to claim 5 wherein said    sample is aqueous, biological, environmental or a food product.-   2. A method for capturing BoNT/B from a sample, said method    comprising contacting said sample with the monoclonal of claim 1 or    3 and isolating the complex formed between the BoNT/A in the sample    and the monoclonal antibody.

What is claimed is:
 1. An isolated and purified monoclonal antibodyproduced by the continuous hybridoma cell line having deposit accessionnumber ATCC PTA-11872.
 2. A composition comprising the monoclonalantibody of claim 1.